Functional centromeres determine the activation time of pericentric origins of DNA replication in Saccharomyces cerevisiae.

Molecular and Cellular Biology Program, University of Washington, Seattle, Washington, United States of America.

Abstract

The centromeric regions of all Saccharomyces cerevisiae chromosomes are found in early replicating domains, a property conserved among centromeres in fungi and some higher eukaryotes. Surprisingly, little is known about the biological significance or the mechanism of early centromere replication; however, the extensive conservation suggests that it is important for chromosome maintenance. Do centromeres ensure their early replication by promoting early activation of nearby origins, or have they migrated over evolutionary time to reside in early replicating regions? In Candida albicans, a neocentromere contains an early firing origin, supporting the first hypothesis but not addressing whether the new origin is intrinsically early firing or whether the centromere influences replication time. Because the activation time of individual origins is not an intrinsic property of S. cerevisiae origins, but is influenced by surrounding sequences, we sought to test the hypothesis that centromeres influence replication time by moving a centromere to a late replication domain. We used a modified Meselson-Stahl density transfer assay to measure the kinetics of replication for regions of chromosome XIV in which either the functional centromere or a point-mutated version had been moved near origins that reside in a late replication region. We show that a functional centromere acts in cis over a distance as great as 19 kb to advance the initiation time of origins. Our results constitute a direct link between establishment of the kinetochore and the replication initiation machinery, and suggest that the proposed higher-order structure of the pericentric chromatin influences replication initiation.

A schematic diagram of chromosome XIV in wild-type (WT) and rearranged strains.

(A) In the WT strain, the BglII restriction site in MET2 is located 8.5 kb to the left of ARS1410. Centromere XIV resides in its endogenous position located 6.8 kb to the left of ARS1426. (B) In the rearranged strain the endogenous centromere was replaced with a URA3 selectable marker while a functional centromere was integrated along with LEU2 (open box) into the MET2 locus such that the centromere was positioned ∼11.5 kb from ARS1410. The white and black arrowhead above each centromere indicates the direction of the centromere DNA elements CDEI, CDEII, CDEIII.

Replication time of native and relocated centromeres on chromosome XIV.

(A) Cartoon depiction of experimental setup. Cells were grown in medium containing heavy carbon (13C) and nitrogen (15N) isotopes. Upon genome saturation with the heavy isotopes, cells were arrested by the addition of alpha factor and released synchronously in medium containing light carbon (12C) and nitrogen (14N) isotopes. The cells were then collected over the next 140 minutes and their DNA was extracted, digested with EcoRI, and separated via ultra centrifugation in cesium chloride gradients such that unreplicated DNA resides lower in the gradient than newly replicated DNA. DNA samples were then collected and analyzed through drip fractionation. (B) S phase progression of WT (left) and rearranged (right) cells as measured by flow cytometry. Cells from both strains entered S-phase by 40 minutes and achieved 2C DNA content by 140 minutes as indicated by the peak shift from 1C to 2C DNA content. (C) Replication kinetic curves for met2 or MET2, ARS1410, and ARS1426 in WT (top panel) and rearranged (bottom panel) cells. The kinetic curves for ARS306 and R11 are shown as dashed and dotted lines, respectively. Trep is the time of half-maximal replication for each locus (see ). (D) Replication indices for met2 or MET2 (green), ARS1410 (blue), and ARS1426 (magenta) in WT (solid diamonds) and rearranged (empty diamonds) strains. ARS306 (black dashed arrow) and R11 (black dotted arrow) were used as early and late timing standards, respectively. In the WT strain, MET2, ARS1410, and ARS1426 had replication indices of 0.87, 0.77, and 0.16 respectively. In the rearranged strain, met2, ARS1410, and ARS1426 had replication indices of 0.24, 0.23, and 0.79, respectively. Direction of the black arrows indicates the direction of the shift in replication index for each locus between WT and rearranged strains.

DNA fragments containing a functional origin are detected as a bubble arc (depicted by the bubble fragment) while fragments that are passively replicated are detected as a Y-arc (depicted as a Y shaped fragment). (A) ARS1410 and ARS1426 are functional origins in the WT and rearranged strains. (B) 2D gel analysis of the MET2 or met2 locus in the WT and the rearranged strains. WT DNA was digested with the restriction enzyme StuI giving a fragment of 3.96 kb centered on MET2 (grey arrow). Rearranged DNA was double digested with XbaI and BsrBI resulting in a 4.36 kb fragment harboring most of met2, the integrated centromere (black circle), and the LEU2 marker (white rectangle). The absence of a bubble arc when probed for the 3′ end of MET2 and met2 (hashed rectangle) indicates that an origin is not present on either DNA fragment. The centromere in the rearranged construct was detected as a pause site (black arrowhead) visualized as a dot of relatively increased intensity on the descending Y-arc.

(A) Cartoon depiction of chromosome XIV with a non-functional centromere (white circle) integrated at MET2. Chromosome XIV of WT cells was modified such that MET2 was disrupted with the same sequence used to disrupt MET2 in the rearranged strain (see ) except that the centromere was made inactive by mutating the essential CDEIII domain. This chromosome is maintained through its wild type centromere at the endogenous location (black circle). (B) Flow cytometry of cells with a non-functional centromere in the MET2 locus. Similar to the WT strain (see ), cells from this cell line enter S-phase by 40 minutes and achieved 2C DNA content by 140 minutes. (C) Replication kinetic curves for met2::cen7 and ARS1410. As observed in the WT strain, the replication curves for met2::cen7 (green) and ARS1410 (blue) are positioned more closely to that of R11 (dotted line) than ARS306 (dashed line) (compare to ). D) Replication indices for met2:cen7 (green diamond) and ARS1410 (blue diamond). RIs of ARS306 and R11 are indicated by black dashed and dotted arrows, respectively. met2:cen7 and ARS1410 had RIs of 0.81 and 0.74, respectively. (E) 2D gel analysis of ARS1410. Presence of a bubble arc (black arrow) for ARS1410 in the strain in which the non-functional centromere was integrated at MET2 (compare with ) indicates that ARS1410 is a functional origin in this strain.

(A) Replication kinetic profiles of chromosome XIV in WT (top) and rearranged (bottom) strains. Percent replication was monitored across chromosome XIV at 40 (magenta), 45 (orange), 55 (green), and 65 (blue) minutes following release from alpha factor arrest. When the native centromere (yellow circle) is present near ARS1426, a prominent peak is seen in the 40 and 45 minute time samples. In this strain, the peak at ARS1410 is shallow in the 40 and 45 minute samples. When the centromere is repositioned (orange circle) near ARS1410 in the rearranged strain, both the time of appearance and the prominence of the peaks at ARS1410 and ARS1426 are inverted with respect to the WT strain. See and and and for all chromosomes. (B) Z-score plots of chromosome XIV in WT (black) and rearranged (blue) strains. Replication kinetic profiles from the 40 minute sample were normalized by converting percent replication values to Z-score values (see ). Genomic loci corresponding to ARS1410 and ARS1426 show significant differences in Z-scores. ARS1424 is the next closest active origin to the endogenous centromere residing ∼19 kb to the left. See , , and and for all chromosomes and the 45- and 65-minute samples.

(A) Z-score plot of chromosome XV in WT (black) and rearranged (blue) strains. ARS1531 displayed a difference of Z-score values at least as large as that seen for ARS1410 and ARS1426 (see ). See for all chromosomes. (B) 2D gel analysis of ARS1531 in the WT (left), the rearranged strain used in microarray analysis (middle), and the rearranged strain used for prior slot blot analysis (right). DNA from all three strains was digested with NcoI and BglII to give a 3.18 kb fragment harboring ARS1531 and then subjected to 2D gel analysis. The presence of a bubble arc in the WT (black arrow) indicates that ARS1531 is a functional origin in this strain. The presence of a bubble arc in one of the two rearranged strains confirms that the absence of origin activity in rearranged A (used in microarray analysis) is not due to relocation of the centromere on chromosome XIV. Below each 2D gel image is the sequence for the WT or mutant (red) ACS.

(A) Kinetochore/microtubule interaction orients the centromere and pericentric DNA near the microtubule organizing center (MTOC) where there is an enrichment of replication initiation factors. (B) Tension exerted by the kinetochore/microtubule interaction induces an altered chromatin structure of pericentric DNA that provides accessibility of embedded origins to initiation factors. (C) Kinetochore proteins interact directly (or indirectly) with origin initiation factors recruiting them to nearby origins. (D) The organization of pericentric DNA into the C-loop orients origins within the C-loop to the periphery of the chromatin mass increasing their accessibility to initiation factors.